Partial characterization of guard-cell phosphoenolpyruvate carboxylase: kinetic datum collection in real time from single-cell activities.
Partial characterization of guard-cell phosphoenolpyruvate carboxylase: kinetic datum collection in real time from single-cell activities.
复制标题
保卫细胞磷酸烯醇丙酮酸羧化酶的部分表征：从单细胞活动实时收集动力学数据。
DOI：
10.1016/0003-9861(90)90530-c
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发表时间：
1990
影响因子：
3.9
通讯作者：
W. H. Outlaw
中科院分区：
文献类型：
--
作者：
M. C. Tarczynski;W. H. Outlaw
蚕豆气孔保卫细胞磷酸烯醇式丙酮酸羧化酶（PEPC）的最大速率和Km（PEP·Mg）。的pH值，苹果酸的存在下，和保卫细胞的生理状态的函数。这些测量的生物化学原理是：（a）保卫细胞大量质子排出，这是驱动气孔运动的主要事件，据推测使细胞碱化;（B）保卫细胞苹果酸浓度在气孔打开时增加几倍，苹果酸通常是PEPC的抑制剂，影响一些PEPC的寡聚状态;保卫细胞PEPC在气孔开放过程中的表观活力比其它生理状态下的明显增强。由于存在特定形式的PEPC的细胞特异性表达和不稳定的可逆的翻译后修饰（其在动力学上表现为不同的生理状态同种型）的先例，因此在将单个气孔复合物直接加入到测定混合物的微滴中时开始单独的测定。从在处理前、处理中或处理后冷冻淬灭的冻干叶组织中解剖气孔复合体（每个复合体由一对质量为6 × 10− 9 g的保卫细胞组成），以打开气孔。VmaxnorKm（PEP·Mg）与提取酶的组织的生理状态无关。然而，Km（PEP·Mg）在pH 8.5时比在pH 7.0时低>4倍。苹果酸盐抑制在两种pH下都是竞争性的，但在较低pH下抑制大于3倍。这些数据表明，在所研究的范围内，pH和苹果酸盐的组合效应可以产生约24倍的酶速度变化。因此，结果是一致的解释，即保卫细胞PEPC是由细胞质化学环境，而不是由生理状态异构体之间的交替。
Maximum velocity andKm(PEP·Mg)of phosphoenolpyruvate carboxylase (PEPC) from stomatal guard cells ofVicia fabaL. were determined as a function of pH, presence of malate, and physiological state of guard cells. The biochemical rationale for these measurements is that (a) massive proton extrusion from guard cells, the primary event that drives stomatal movements, has been speculated to alkalinize the cell; (b) guard-cell malate concentration increases severalfold on stomatal opening, and malate, generally an inhibitor of PEPC's, affects the oligomeric state of some PEPC's; and (c) the apparentin vivoactivity of guard-cell PEPC is greatly enhanced during stomatal opening, compared with that of other physiological states of these cells. As there are precedents for cell-specific expression of particular forms of PEPC and for labile reversible, posttranslational modifications (which are manifested kinetically as distinct physiological-state isoforms), individual assays were initiated on the addition of a single stomatal complex directly to a microdroplet of assay cocktail. The stomatal complexes (each of which comprises a pair of guard cells having a mass of 6 × 10−9g) were dissected from lyophilized leaf tissue that had been freeze-quenched either before, during, or after a treatment to open stomata.Vmaxat pH 7.0 was not significantly different from that at pH 8.5. NeitherVmaxnorKm(PEP·Mg)was distinguished on the basis of the physiological state of the tissue from which the enzyme was extracted. However,Km(PEP·Mg)was >4 × lower at pH 8.5 than at pH 7.0. Malate inhibition was competitive at both pH's, but inhibition was >3 × greater at the lower pH. These data indicate that the combined effects of pH and malate over the range studied can produce changes in enzyme velocity of approximately 24-fold. Thus, the results are consistent with an interpretation that guard-cell PEPC is regulated by the cytoplasmic chemical environment and not by alternations between physiological-state isoforms.